Proof-of-principle: targeted childhood leukemia prevention

is the most common cause of disease-related childhood mortality in developed

Cancer is the most common cause of disease-related childhood mortality in developed countries, with B-cell acute lymphoblastic leukemia (B-ALL) representing the most frequent. A salient characteristic underlying some cases of childhood B-ALL is the presence of congenital mutations (either inherited or de novo) that are compatible with normal lymphocyte development, but lead to the appearance of a silent population of preleukemic cells that acquire additional genetic mutations and ultimately progress to full-blown B-ALL. The precise mechanisms underlying malignant transformation have been difficult to ascertain due to their development in otherwise healthy-appearing children. Notably, several decades ago, delayed exposure to common infections was postulated as a triggering factor leading to the appearance of the secondary genetic lesions required for B-ALL to develop [1].
Using mouse models genetically engineered to carry leukemia-predisposing germline mutations found in human B-ALL patients, several recent studies have demonstrated the existence of this infection-triggered process in the progression towards B-ALL, providing evidence that different types of immune stress can activate the clonal evolution of preleukemic precursors [2][3][4]. One key aspect identified through these studies is that the transforming effect of infection does not result from the outgrowth of preleukemic clones already carrying one or more second hits; instead, the effect of infection-induced immune stress is to trigger the actual appearance of these second mutations, thereby directly causing progression to B-ALL [1]. Under these premises, it becomes conceivable that one could prevent the development of B-ALL by eliminating the preleukemic clone [1][2][3][4][5]. However, one would have to find a way to specifically target these preleukemic cells. Recently, a mouse model recapitulating the phenotype of a leukemia-predisposition syndrome has allowed us to carry out a proof-of-principle experiment to achieve this very goal. are compatible with a normal hematopoietic development. However, immune stressors, such as exposure to common mouse pathogens after transfer to a non-SPF animal facility, trigger progression to B-ALL (red cells) through the appearance of secondary mutations affecting the Jak/Stat pathway. Transient treatment of Pax5 +/− mice with the Jak1/2 inhibitor ruxolitinib destroys these progression-prone preleukemic cells and significantly reduces the risk of leukemia development. These results demonstrate that there is a window of opportunity in early postnatal life during which preventing the progression to B-ALL in predisposed children might be possible. Mo: months-old.
Children carrying heterozygous mutations affecting the B-cell master regulator gene PAX5 are predisposed to develop B-ALL; similarly, 25% of heterozygous Pax5 +/− mice develop leukemia, but only after experiencing an immune stress, such as exposure to infection [2][3][4]. Furthermore, the B-leukemias that appear in Pax5 +/− animals acquire similar mutations as observed in the leukemic blasts from humans harboring pathogenic PAX5 variants, including activating mutations affecting the Janus Kinases (JAKs) [2]. We previously demonstrated that, in Pax5 +/− mice, early B cell precursors (pro-B cells) are very dependent upon interleukin-7 (IL-7) for their survival. Further, blockade of IL-7 signaling by treatment with the JAK1/2 inhibitor ruxolitinib led to apoptosis of Pax5 +/− pre-leukemic B cells in vitro [2]. Taking advantage of this knowledge, we have recently used Pax5 +/− mice to evaluate whether in vivo treatment with ruxolitinib early in life will kill preleukemic cells and, therefore, prevent the development of acute leukemia [6]. Pharmacokinetic studies were performed to determine appropriate doses and then wild-type (WT) and Pax5 +/− mice were fed with ruxolitinib in their chow. Mirroring the in vitro results indicating high dependence on IL-7 signaling, treatment with ruxolitinib mainly led to the disappearance of B-cell progenitors in Pax5 +/− , but not in WT, animals [6].
Therefore, in the next experiment, both experimental Pax5 +/− and control WT animals were fed with ruxolitinib containing chow for 14 or 28 days, starting from the moment they were transferred from a specific-pathogenfree animal house (SPF, where they had been born and weaned) to a conventional facility, where they were exposed to common mouse pathogens (Figure 1). The animals treated with ruxolitinib for the longer period (28 days) exhibited a significant 90% reduction in the incidence of B-ALL when compared to untreated mice, or to animals treated only for 14 days [6].
Ultra-deep sequencing studies of Pax5 +/− mice had previously shown that the activating mutations affecting Jak are only detected once the animals already present with the full-blown B-ALL, and never during the preleukemic period [2]. Furthermore, when a constitutively activated Jak3 V670A transgene is added to the Pax5 +/− genotype, the animals develop B-ALL immediately and without the need of exposure to any immune stress. Therefore, it is unlikely that the reduction of the incidence of acute leukemia after treatment with a Jak inhibitor for 28 days is due to the selective death of Pax5 +/− progenitors already harboring leukemogenic mutations in this pathway. On the contrary, one can conclude that ruxolitinib is acting by eliminating predisposed preleukemic B cells before the second hit leading to B-ALL arises in them (Figure 1).
These data show that ruxolitinib-mediated inhibition of the JAK-STAT pathway in predisposed Pax5 +/− animals prevents the development of leukemia, and suggest that an analogous approach could be used to prevent progression to B-ALL in children at increased genetic risk, either because they are carriers of PAX5 or due to other predisposing germline mutations. Importantly, since the beneficial action of ruxolitinib in predisposed animals could be achieved following transient treatment with the drug, one can extrapolate that any future treatment of predisposed children need not be sustained for a long period, but just administered during a specific time window during their childhood.
It is becoming increasingly clear that the existence of latent pretumoral cells is common to many types of both hematologic and solid cancers [7]; therefore, the concept described here could be considered a proofof-principle strategy for the development of similar prophylactic approaches to prevent the progression of other malignancies. Still, some aspects remain unclear. For example, why do the majority of genetically predisposed animals (and most genetically predisposed children) not develop leukemia and stay healthy? In addition, what are the mechanisms by which environmental factors such as infection promote the acquisition of secondary mutations leading to malignant progression of preleukemic cells? These and other important questions still need to be answered if we are to fully understand and avert the appearance of B-ALL.

Author contributions
The authors contributed equally to all aspects of the article.

ACKNOWLEDGMENTS
We are indebted to all members of our groups for useful discussions and for their critical reading of the manuscript. Research at C. Cobaleda's laboratory was partially supported by Ministerio de Ciencia e Innovación/AEI/FEDER (PID2021-122787OB-I00), and a Research Contract with the "Fundación Síndrome de Wolf-Hirschhorn o 4p-". Institutional grants from the "Fundación Ramón Areces" and "Banco de Santander" to the CBMSO are also acknowledged. Research in C. Vicente-Dueñas group has been funded by Instituto de Salud Carlos III through the project "PI22/00379 and by a "Miguel Servet Grant" [CPII19/00024 -AES 2017-2020; co-funded by European Regional Development Fund (ERDF)/European Social Fund (ESF) "A way to make Europe"/"Investing in your future"]. Kim Nichols receives funding from the American Lebanese Syrian Associated Charities (ALSAC) and R01CA241452 from the National Cancer Institute Research in ISG group is partially supported by FEDER and by RTI2018-093314-B-I00 MCIU/AEI/FEDER, UE; by PID2021-122185OB-I00 MCIU/AEI/FEDER, UE, and by Junta de Castilla y León (UIC-017, and CSI144P20). M. Ramírez-Orellana and I. Sánchez-García have been supported by the Fundacion Unoentrecienmil (CUNINA project). C. Cobaleda, M. Ramírez-Orellana, and I. Sánchez-García have been supported by the Fundación Científica de la Asociación Española contra el Cáncer (PRYCO211305SANC).

CONFLICTS OF INTEREST
Dr. Nichols reports research support from Incyte. A.W. is a full-time employee of Novartis Pharma AG. The remaining authors declare no competing financial interests.